Lecture Notes (Before Midterm 2) PDF

Lecture Notes (Before Midterm 2)  Oct 1 ,...

Lecture Notes (Before Midterm 2)  Oct 1 , 2024 Protein Structure Lehn Chapt 4 General Themes : proteins ability to Change Shape make this 1) 3D fold determined of I structure not as by Seq easy as it seems 2) protein structures are dynamic (conformations proteins quite flexible exisit in a few closely related structures of similar energy are generally nature is important for function parts of proteins can move w/ small dynamic E barrier. lavoid e g. loop in active. site to keep water out competing interactions / binding 3) 3D fold. is stab. by many weak forces and interactions H-bonds Critical for 2 Structure Force (polar) Salt-bridges drive a Hydrophobic effect Critical to folded State Entropically 4) Although dynamic protein function depends on 3D fold , Disruption of 3D folding function is disrupted e g. misfolded proteins is characteristic. to Alzeihmers or Parkinson disease Peptide Bond peptide bond : rigid peptide bond : Gatoms of Peptide Ri bond Casino - Coz Ca Con C2-Co2 peptide group lie in a single plane - Ca Ca 2 g Rz Resonance of peptide bond results in partial double bond character and ' have Amino acid residue. no rotation about C-N bond Can" -Coz distribution of e- create dipole polar bond : Restriction on Rotation in polypeptide i R, N(pSi) N-CC) ①(phi) and Y (psi) rotation could result many possible backbone Ro-- in - - Col conformations R2 O(phi) Steric hindrance between R group and Carbonyl of peptide results in restrict. Conformations Ability of peptide bond O and. NH partial CFN bond character results in Certain conformations favoured (particular philpsi angles antiparallel B-sheets righttwistedShethe possible Ramachandran Plots su Lenn 4 2 Collagen triple helix tranas. Plot of angles collide making them not Endices certain left-handed various philpsiangles a " yo""!, regions wi small amount of steric hindrance loverlap allowed not within Common 2 structures regions regions wi no steric overlap favoured conformations (2 structure hard to have oo = left-hand helices not c helices (only few res. in structure , /, [ C-helices- wo collisions right-handed c Angles in "not allowed " forbidden region is due to strain on protein helices - 180 180 180 ① S -  can measure particular a a/peptide and plot phi and psi angles to look at regions angles plotted Other conclusions from plot lay a particular res in. particular region is not definite of 2 Structure ~ 25 of conformational space (philpsi) is accessible : -15 2 Amino Acid Exceptions is "forbidden" Proline 1 Glycine = RH , no chirality results in Dangles restricted by ring structure added flexibility and : Can occupy * - 4x4- more phil psi space plotted angles appear in more are as very small/limit. range of rotation for psi values Cisvs Trans. Peptide Bond Trans (most common/favourable Cis Ri 2 R groups are on p , - 2R groups are on - - - Cc-carbon opposite sides o f - the same side of Rz peptide bond peptide bond Cis conformation is energetically unfar due to steric clashes. Exception When R2 is Pro Trans Conformer also energet unfar) Cisnot more far. :. Proline Peptide Bond (cis vs trans. Trans VS. Cis ↓ " "D -" Both Conformation have steric clashing - - J Diff. energy of Cis-and trans proline is much less than other a a due to rigidity. Trans-also unfar. allowing lis-proline to be formed but never see cis conformer of other aa.. 2 structure 2 main structures cc-helix and : B-Strands/ Sheets Net dipole electric dipole of : peptide bond is loops and turns also discussed (not rily 20 Structure transmitted along CC-helix through C-helices : Spring , Spiral Shape intrachain H-bonds overall helix dipole DDDDO Parallel to helix axis and occurs from rest H-bonding : ity coo G Continuous set of a a res.... res. 5 , 2 6 3T etc , , / can be is H-bonded Within the helix every res. distorted Exceptions : i it pitch 5 4 lturn =. 1) first 4 res of cc-helix do not donate a. H-bond acceptor is 4 distance between res before donor H-bond via NH group (within helix) ea turn.. dt Side chains be positioned to form H bonds 3 Gres Hurn may.. NHat  2) last 4 res do not accept an H bond via. Carbonyl group (within helix) keeps helix more 3) Other H-bonding groups form H-bonds at end of helices (capping) Stable Res within helix have Similar I , values. , 4 due to repetitive geometry 4-3 N-term. Fig Rgroups in res. project outwards perpendicular to helix axis Affects cc-helix function C-term. Helical wheel WI 3 6 res/ turn. in helical Wheel 1000 : per res. R-groups on each side of whee R, Ro properties affect cc-helix properties R4 3400 Rs If : R , Rs Ro Ry are hydrophobic amphiphatic helix. polar and non polar opposing 48 , , 300 and face away from ag. environ. faces (common Ry Ra Ro Re are hydrophilic , , , Important andCommon feature for folding protein 100 R, 240 R2 If res. 1 and it 4 are res that into 3D. Shape 200 148 far )/ I can stab. helix R Ro interact. vos e g Glu. and Lys are res. lands SaltbridgeStab. interaction Features NHz The probability ofCertain a. a. are preferred in cc-helices while others are uncommon "propensity" Ala is most common. (larger R groups may interact unfav ). Gly and Pro are not common Glycine Proline important for H-bonds DandY angles are not restrict. found in Bulky and H is missing in NH of peptide bond loops and turns as they require&and 4 flexible angles Cannot be in helix except at N-term ① is restrict. BulkyRgroup Carbonyl0 & N-term. vs Other a. a.. has (in place of H. Ri can accept 1 H-bonds Xx Pro. "helix breaker" Causes Kink in bond and... no continuous longer a Stretch of a a. res.  Bstructure B-strands and B-sheets : & Yandd are closer to 180 (compared to Cc-helix) y Results in extended backbone wi repetitive & and 4 values ① Backbone described as "zig-zag Shape" B-sheets form from multiple B-strands continuous set of aa. res Not continuous set of a a. res Lor more B-sheets form a B-strand Stab. by H-bonds between strands and roughly perpendicular to backbone laxis B-Strands Can be parallel (term. in Same direction) , antiparallel term. in opp directions). , or a mix parallel B-Strands antiparallel B-strands Antiparallel Parallel R -i R of - R groups on same side - B-sheet H-bonds : at a slight Rz H-bonds perpend. to B-strands Bangle : Rs R -- - Parallel to eachother - M M Rs Ro Ry R groups on adjacent strands but same position on the sheet R-groups orientated on same side of plane are oriented in the same direction Amphipathic B-Sheets : have I hydrophobic andI hydrophilic faces B-Sheet (continued RR RR RR Zigzagbackbone could be amphipathic if 2 R groups have diff. polarity : I hydrophobic face I mainly non polar res. and other RR RR face is hydrophilic (mainly polar res. flatB-sheets are rare would expect hydrophobic face to be buried (away from ag. environment Most B-sheets are twisted (most common) each strand at a slight angle to each other Have atleast 2B-strands (held together w/H-bond.) Can be parallel antiparallel , or mixed parallel antiparallel mixed Linkages between 2 Structure Many ways it can occur follow same "rules" i the Linkers fold back to allow 2 Linker regions : no 2 structure (no repetitive and Y angles structures to fold. can be loops or turns  2 Structure Folding to Form B-Sheets Antiparallel B-Sheet Parallel B-Sheet Loops Often flexible ⑤ N C " C No repetitive and Yangles May not need as many No regular H-bonding loop res. Shorter In B-sheet the res. that form Strands do not need to be Gly and Pro. Effects Regions eeg. in Linker close in sea v 2 B-Strands Glycine more important than achiral C connected through R H small and the only = : B-turns min res.. to connect B-strands and max res to do.. compacted non chiral a G.. more and Y values 4 a a res that connect antiparallel.. B-strands Less restrict. On O and 4 Rigid and compact N Allows for tightening of backbone structure unlike loops : Proline 2 and 3rd res. Often Gly and /or. Pro. Puts Kink in backbone 1st and 4th res Often Pro. have H-bond. :. not critical Phi(0) angle restrict. NH in peptide bond lacks Exam Q. Note 12 ways It in amino group found in other regions I. B-Sheet alternating : polar and n. p a. a res... Cannot exist in cc-helices or B-sheets and Gly/Pro in 4 Seq. res. Adds to rigidity of turns. 2 Specific sea(polar and n p.. resl give helical Sequent Analysis Wheel May be able to predict B-Strands Gly and Pro (B-turns) may predict antiparallel set of B-Strands Especially if sea. pattern suggest amipathic B-sheet alternating polar and non polar res. Motifs Multiple 2 elements come together to form 3D Shape (subset of whole structure necessarily a complete Not functional Structure "Building blocks" g B-CC-B Side Chains e Cc-helix Connecting 2 B-strands :.. Hydrophilic res. * NHydrophobic side cana C rotate : end- on view hydrophobic effect allows structures to fit together Can build large structures we motifs : Both the B-Sheet and cc-helix are amphipathic Can be of multiple same or multiple e. g (cc Blo Structure. - I ******** diff motifs 2. Doesn't appear stable but the 2 ends have H-bondingCapacity -C can fold to form B-barrel  Bearre rawenvanhelices H all B-Strands H-bond. together bond internal water and form pores Small barrels have no space due to R groups inward pointing Domains Region of Seg 11. structure that folds into a 3D unit Proteins can have lor more domains If they are close Like 2 Structures domains are linked by loops , or other regions wh no 2 Structure Ca a.. linking motif 3 structure Folded functional structure which can be : loops can be quite long /no repetitive & , allcc-helices land loops) and N values 2 all B-Structures(and loops and turns) only for B-strands (a subset of loops 3 a mix of c and B Structure (and loops and turns) Energetic Contribution Hydrophobic effect burying of hydrophobic : R- groups major energetic contribution to folding H-bonds help define : and maintain 2 Structure elements Ionic interactions Salt bridges/electrostatic : interaction Disulfide bonds covalent bond gives extra stab. to protein : not in all proteins : Common in extracellular loxidizing environments Only formed after protein is folded 3 Structure WI more than I domain (s) can form close contact wi each other Domains connected by extended linkers "beads on a & contacts exclude String" E3 water may require contact for OR Often each domain Stab / function. has specific function : may not rea. connected by flexible linkage for stab. linkers domains may have e g A2-domain.. protein , each may complimentary functions have specific function functional Synergy binding to Carbohydrate lig and catalytic activity 4 Structure 2 or more polypeptide that makes up proteins terms used monomer , protomer , Subunit Synonoms Mainly held together via hydrophobic effect Entropic effect needed to free water has other interactions (electrostat and. H-bonding Some have inter subunit disulfide bonds (extracellular proteins in Oxi. Conditions) Disulfide bonds can be inter-or intra- Homomultimer and heteromultimer Homomultimer Heteromultimer A A CC B B CC Classes ofStructure 1) Fibrous proteins 2) Globular proteins 3) Intrinsically disordered protein (IDP) doesn't have ordered structure Fibrous Protein Lehn 4 3. Usually only have I type of 20 structure all helices or B-sheets In Chelix One : long helix (29 Do not fold into 30 structure.. these rise to extended structures give Have many hydrophobic res. important in folding to 4 structure Generally not water-soluble Functions : Their 40 structures pack to form a higher-ordered structure Support structures connective tissue 'supramolecular structure' forms an array of proteins · Protective Skin hair nails horns , , , May be rigid or somewhat flexible Force muscles Structure (rigid us flexible) relates to their function * focus for course 1) C Keratin hairs nails and horns Main component of , , Family of proteins : Some similarities give rise to 2 structures 1 structure variations in seq. give rise to variations Heptad repeat 17 a a.. res ). repeating in structural properties variation in function in structure labcdefgln Makes up sea WI. little gaps hydrophobic res. Itypically  2 Structure CC-helical but distorted compared to "ideal cc-helix" Dand Y angles slightly diff. results in axis of helix no longer straight slight change in pitch and... of res in turn and the. helical axis changes CC-Keratin "Ideal helix". 5 reslturn 3. 6 reslturn 3. A pitch 5. 4 A pitch 5 Ix18 i (not Slunit but used commonly Fewer res and slightly 22222. smaller pitch ⑳88888 , The helical axis forms a left handed twist (supertwisted R-handed spiral twisted in left handed fashion 3 structure Identical to 2 structure 4 Structure Dimer of L-handed twisted cc-helices 'Coiled Coil' General term not specific to cc-helix 2 c helices Nu C Appears like long protein wrapped around N -X- due to extended structure each other in a l-hand. 4507 twist to form structure 300 res. ~ 88 turns of a helix HelicalWheel (only Co Shown Dimers join together in supramolecular compound Electrostatic interact. may be possible if e and & g are opp Charged res. 9. C D e g Asp Glu Salt bridges.. + d A Hydrophobic interface hydrophobic effect : f f holds subunits together A d D C C 9 Important to align helices Variations in seq. of heptad repeat at positions other than a andd results in different types of Keratin "b" "C" and "f" res Can interact w/ other cc-Keratin molecules , ,. creates Supramolecular structures Sea. differences result in functional differences.g e. hair some flexibility disulfide bonds Some : cys res. form intramole disulfide bonds. various changes occur I res On same side is not. possible which may affect these Hair perms: I Cys-Cys bonds are red. frees them ( flexibility c Curl hair around rod 3 Reoxidize hair to form new cys-cys disulfide bonds holds curl shape nails more rigid proportion of cys res and... more covalent cross links between diff cc-Keratin molecules. Makes more rigid / tougher structure 2) Collagen Found in connective tissue, tendons , cartilage and bone more rigid than Cc-helix Roles in structural integrity Has a lower level of flex. VS Cc-Keratin 1 structure often Pro. or 40H-Pro. 3 res repeat. : Gly-X-Y a a.. composition : 35 % Gly , 20 % Pro. , 11% Ala almost always often Pro. at X or Y position may be OH 4- OH Pro Post translationally 2 Structure Due to numerous Pro. not as common structure - A N -mod by add polar. Forms L-hand helix w/33 rest turn wh a. pitch group that can H-bond 9. 47 Pro. bangle Extended Structure (0 4--75 C 145 ) ° % , , In B-Structure region 3 structure No folding back on itself and... is equal to 2 structure 4 Structure Construction 3 hand. helices wrapped around each other forms a Rhand. Superhelix requires other Individual L-hand Collagen-like helix is not stable. factors which are Requires triple Superhelix to maintain structure removed No H bonds within each polypeptide : triple helix structure is stab. by inter-chain H-bonds and hydrophobic interact. Close packing of polypeptide enabled by Gly's small Size Textbook refers Gly NH can donate an H-bond to the Pro C O. = group to as cc-chains on adjacent polypeptide chain NO OH group :: Can only act as H-acceptor  Dimensions 1000 a A very extended little elasticity and If polymer extended cannot ~.. : - 300 repeats 3000 long great tensile strength Stretch Supramolecular Structure H-bonds and covalent crosslinks 4-OH-Pro can form H-bonding between triple helices points outwards from helices Pro 4-OH-Pro Vitamin C deficiencies prolylhydroxylase scurry wo its unable to 3) Fibron (Spider silk) form interact. in 1 structure 2 Structure supramole. Structure Rich in Ala and Gly (2 smallestaal Beheets connective tissue 3 structure starts to break down BSheets not folded into globular shapee..= 2 structure Idisruption of conn. 4 Structure tissue B-Strands B-sheets Alternating Ala and Gly res B-sheets stack together. H-bonds between B-strands to form B-Sheets ChaCHaCHa CHaCHaCHa CH3CHz CHz Stacking of B-sheets is a result of hydrophobic interact. B-sheets drawn as repeats I sheets although not necessary wI Gly HHHHHH interact : 3 57 H H H H H. HH HH Gly pack together as Ala align Sheets stack underneath Interdigitation of Ala and Gly Side chains : Ala has held CH3CHzCHz CH3CHzCH3 allows for close further apart 50 : CH3CHzCHz CHaCHzCHz CHaCHzCH3 packing of sheets flexibility can shift and still hold sheets together allows for flexibility of stacked B-sheets HHHHHH B-structure is extended and cannot be stretched Still has a level of flexibility  Summary CcKeratin Collagen Fibroin 2 c helices from 3 hand. helices that B-strands that form B-sheets ↳hand Supercoil. form R-hand Supercoil. ("Coiled coil" Heptad repeat : 'a' and 'd' positions G -X Y - - A-G- (not perfect) are hydrophobic Not extended and : Cannot Extended : no ability Extended.: Cannot stretch Stretch to stretch Variable degree of toughness Rigid and has many crosslinks Flexible due to lack of covalent depends on of disulfides between collagen molecules Crosslinks All of these proteins exisit in "families" variations in a.a. sea can affect their structure and function Many proteins have structures making up whole structures Heptad repeat may be present but the protein is not a fibrous chelix , May hold dimers together Globular Proteins Folding Process Lehn 4 4. Difficult to observe : can be very fast may start as protein is synthesized on ribosome e Unfolding and refolding are studied Not a random process not all $ and Y possibilities are explored would take too long Methods 1 Heat Abrupt loss of structure 108 Cooperative Process Cooperativity due to large of weak non-covalent 50 interactions that stabilize the 3D fold native Tm : wI more salt-bridges and better, more tightly packed hydrophobic core T( C) O Tm  2 Pt ExtremepH / for 12) Disrupts salt-bridges and H-bonds Adds a net charge on R groups results in electrostatic interactions 3 Chaotropic Agents (urea gaunidium salt) 8M GM Forms H-bonds to proteins disrupts 2 structure Stabilize and solubize unfolded state Disrupts hydrophobic core Refolding Experiments Raise temperature and slowly cool often irrevers. due to aggregation of unfolded state Urea used to unfold T Dayisba can be removed slowly by dialysis ermeable membrane : allows passage of small molecules Buffer wourea Anfinsen Experiment Slowly urea inside dialysis bag allows for : refolding Ribonuclease A 124 9 A.. Scys res... 4 disulfides S S Denatured in urea and red. agent added Cys Cys Cys SH HS Cys unfolded and no disulfides Remove Grea Remove red. agent Cys-Cys possibilities keys oxidizes 7x5x3 105 = Remove red. agent I activity corresponds to randomly Callows Oxi ). Remove urea forming I out of 105 possibilities 100 Activity / ~ Activity Conclusions 1) I structure determines the 3D fold Incorrect fold no activity 2) Disulfide bonds often form after the 3D fold is achieved Disulfides can stabilize the folded structure but do not determine it Folding Process Fast (-ms timescale) , Proteins fold to a small of low energy conformations Dynamic nature Similar structures wh low energy barrier between conformations Width represents of conformations Semi Stable unfolded State intermed. E Not necessarily a smooth trajectory Few low Energy Conformers Functional (folded State Different models of folding 1) Formation of 20 Structure first then those rearrange to form 3D fold 2) Collapse to bury hydrophobic res and their 20 structure starts to form. In vivo Folding may begin on ribosome Cellular components to aid folding Chaperones provides environment for : refolding prevents aggregation due to exposed hydrophobic regions Disulfide isomerase assists : w/ correct disulfide formation reducing Cys-Cys refold reoxidizes Peptide protyl cis-trans isomerase : Cat interconversion of cis-trans Pro. Misfolding Diseases Alzheimer's Parkinson's Not clear if they cause the disease , , Huntington yet or a Amyloidases formation of amyloid fibrils Symptom Amyloids Bstructure : stacked layers of B-sheets Mutations of relevent proteins make formation of amyloids more likely Destab the native fold. Stab. misfolded State cleared Alzeimer's by " proteases Amyloids form in brain peptide N-term. amyloid precursor protein (APP) 142 ABI-H2  N Stacked B-strands C form B-sheets C T N Zh Hydrophobic R groups packed together Alzeimers Proteins (amyloid short 42 a a peptide AB142.. : Forms hydrophobic interactions Hydrophobic res Eeeer Repre between Sheets turns connecting B Strands Different example Prions (Infectious Proteins Pathogen Contains no nucleic acid Results in Scrupie Bovine , Spongiform encephalopathy (BSE) mad cow disease Misfolded forms of normal protein (normal function cat misfolding of normally folded proteins. provides scaffold for misfolding Forms amyloid structure Misfold Can occur blo the current 3D fold is only stab. by many weak forces temp affects fold.. Can be stab. and destab molecules that binds to edge of B sheet may stop add of B Sheets : Intrinsically Disordered Proteins (IDP) (or Intrinsic. disorder. proteins lo structure reacts wi aqueous environment disrupts 28 Tend to be rich in charge , polar or Gly Prores ,. low in hydrophobic res. Cannot form hydrophobic core Typically no 20 structure Function Lack of hydrophobic core results in higher flexibility allows for adaptability and functional promiscuity Have a role in regulation (Interact. Wh other proteins e. g. Calpastatin binds to and inhibits several calpain Inabsence of calpain enzyme : much more flexible ribbon-like and forms a structure in presence of enzyme  Role regulation of transcription and translation in Function is regulated by PTM (Post translational modifications polar res expect most interact. through polar interactions : e. g. I Phosphorylation of Ser Thr , , or Tyr X Kinasex P Phosphatase neutral neg. charge 2 Acetylation of Lys acetyltransferase H NH3 N CH3 deacetylase pos Charge neutral/larger. An important regulator of proteins wh charged / polar group 3 Methylation of Lys NH3 NHz CH3 but POS Charge unchanged NHS. bulk is increased CH3 NCH3 CH3 PTMs influence or affect binding of IDP to its targets Structure and Function of Globular Proteins ways to study Structure, Function Gly / Pro conservation likely ILook at sea / sea.. Alignment impliesStructural importance Sea conservation and diff. Implies functional importance can do exp. to a. a. res to see effect on structure and function change. 2) Determine 3D Structure X-ray crystallography NMR : more useful for dynamic properties Useful in determin interact.. Cryo-EM In all cases: aim to get atomic level detail of 3DStructure Often combined wh other exp techniques. (e g. mutagenesis). used as guide for future exp.  3) Functional studies Function implies molecular interacts wh other molecules EnzymeCatalysis Ligand binding Ligand Binding A ligand is any other molecules which forms revers. non covalent interact. Hydrophobic effect H-bonds Electrostatic interactions (salt bridges Must result in a change in function or triggers some other interact. Due to conformational changes (dynamic nature of proteins eg.. "Signalling" Triggers change in function Interactions Reversible and tend to be specific chem. complimentarity Specificity depends on shape and physiochem. properties Ligand bound complex may have : Specificity exists in Spectrum : in high specificity (antibodies wi antigens Hydrophobic interact. Electrostatic interact. more general specificity (histone W/DNA) Ligand fits within protein histone has lots of pos Charge and pl :. interacts wh neg. Charged DNA backbone Binding Affinity Description equilibrium dissociation PL PL constant protien ligand complex Kd PL PL As binding affinity more complex present Kd Measured saturation describes how many binding Spots are filled P Fraction of binding sites filled by lig and PL P fractional saturation Graphical Representation Dissociation Constant Rectangular hyperbola wi asymptope & rearrange. PL PL PL I Kd Kd when 0 5 the igand ligand 2 Kaz is-2x that of Kd , PkaP ,. ↳ Lisequal to Kd and binds wi half 05. - thefinity of L a Ka L Kd , ↳ Kaz L Ka  At equilibrium the free concentration (not bound LC Assume you ligand can measure Rectangular hyperbolashape means : I every 0 5 when. L Kd Both curves should reach max Saturat).. 1) Plot of free ligand : represents saturat , Kd Range (1mM) 10 - 12 M((pM) 3 10 : M ligand weak binding